Agustín Rodríguez

Oxidation, glycoxidation, lipoxidation, nitration, and responses to oxidative stress in the cerebral cortex in Creutzfeldt-Jakob disease

Gel electrophoresis and Western blotting of frontal cortex homogenates have been carried out in sporadic Creutzfeldt-Jakob disease (CJD) cases and age-matched controls to gain understanding of the expression of glycation-end products (AGEs). N-Carboxymethyl-lysine (CML) and N-carboxyethyl-lysine (CEL) were used as markers of glycoxidation; 4-hydroxynonenal (4-HNE) and malondialdehyde-lysine (MDAL) as markers of lipoxidation; and nitrotyrosine (N-tyr) and neuronal, endothelial and inducible nitric oxide synthase (nNOS, eNos and iNos) as markers of protein nitration and as sources of NO production, respectively. Age receptor (RAGE) and Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) expression levels were also examined. The results showed a significant increase in the expression levels of AGE (p<0.05), CEL (p<0.001), RAGE (p<0.05), HNE-modified proteins (p<0.01), nNOS, iNOS and eNOS (p<0.01 and p<0.05, respectively), N-tyr (p<0.05), and SOD1 (p<0.05) and SOD2 (p<0.05). No relationship was observed between PrP genotype, PrP type, PrP burden, and expression levels of oxidative stress markers. The present findings demonstrate oxidative, glycoxidative, lipoxidative and nitrative protein damage, accompanied by increased oxidative responses, in the cerebral cortex in sporadic CJD. These results provide support for the concept that oxidative stress may have important implications in the pathogenesis of prion diseases.

Aquaporin expression in the cerebral cortex is increased at early stages of Alzheimer disease

Abnormalities in the cerebral microvasculature are common in Alzheimer disease (AD). Expression levels of the water channels aquaporin 1 and aquaporin 4 (AQP1, AQP4) were examined in AD cases by gel electrophoresis and Western blotting, and densitometric values normalized with beta-actin were compared with corresponding values in age-matched controls processed in parallel. In addition, samples of cases with Pick disease (PiD) were examined for comparative purposes. A significant increase in the expression levels of AQP1 was observed in AD stage II (following Braak and Braak classification). Individual variations were seen in advanced stages which resulted in non-significant differences between AD stages V-VI and age-matched controls. No differences in AQP1 levels were observed between familial AD cases (FAD, all of them at advanced stages) and corresponding age-matched controls. Immunohistochemistry showed increased AQP1 in astrocytes at early stages of AD. Double-labelling immunofluorescence and confocal microscopy disclosed AQP1 immunoreactivity at the cell surface of astrocytes which were recognized with anti-glial fibrillary acidic protein antibodies. No differences in the levels of AQP4 were observed in AD, FAD and PiD when compared with corresponding controls. These results indicate abnormal expression of AQP1 in astrocytes in AD, and they add support to the idea that abnormal regulation of mechanisms involved in the control of water fluxes occurs at early stages in AD.

Inter-laboratory assessment of PrPSc typing in Creutzfeldt-Jakob disease: a western blot study within the NeuroPrion consortium.

Molecular typing is of considerable importance for the surveillance and epidemiology of human transmissible spongiform encephalopathies (TSEs). It relies on the detection of distinct protease‐resistant prion protein (PrPSc) core fragments that differ in molecular mass and/or glycoform ratio. In this collaborative study, we tested the inter‐laboratory agreement in TSE molecular typing. Sixteen characterized brain specimens from sporadic TSEs and variant Creutzfeldt‐Jakob disease (vCJD) cases were distributed blindly to seven laboratories for molecular characterization by a defined protocol and classification. Agreement between laboratories in the classification of samples was excellent. In particular, there were no differences in the distinction between PrPSc type 1, type 2A, and type 2B with one exception, which eventually was identified as a case with types 1 and 2 co‐occurrence. This shows that the general technique and particular classification system used here are robust and represent a reliable basis for diagnostic and epidemiologic purposes. The subtle further distinction of subtypes among type 1 and type 2 groups requires high‐sensitivity gel electrophoresis protocols that are unsuitable for routine diagnostic needs and must be reserved for research investigations. Further research is necessary on the identification and significance of co‐occurrence of PrPSc types 1 and 2 within one brain.

Clusterin solubility and aggregation in Creutzfeldt-Jakob disease

Prion protein (PrPC) is a glycolipid-anchored cell membrane syaloglycoprotein that localizes in presynaptic membranes. PrP has the property of aggregating into amyloid fibrils and being deposited in the brains in cases with transmissible encephalopathies (TSEs), when PrPC is converted into abnormal protease-resistant PrP (PrPRES). Clusterin is a heterodimeric glycoprotein, the expression of which is enhanced in astrocytes in association with punctate-type PrPRES deposits during TSE progression. In addition, clusterin co-localizes in PrPRES plaques in several human TSEs, including Creutzfeldt-Jakob disease (CJD). Clusterin is up-regulated in the cerebral cortex and cerebellum in CJD as revealed by DNA micro-array technology. Clusterin expression was examined in seven sporadic cases of CJD (codon 129 genotype, PrP type: 4 MM1, 1 MV1, 1 MV2, 1 VV2) and three age-matched controls by immunohistochemistry, Western blotting and solubility. In addition to small punctate clusterin deposition in the neuropil, single- and double-labeling immunohistochemistry disclosed clusterin localization in PrPRES plaques, which predominated in the cerebellum of cases MV1, MV2 and VV2. Moreover, clusterin in plaques, but not punctate clusterin deposits, was resistant to protease digestion, as revealed in tissue sections pre-incubated with proteinase K. Clusterin in CJD, but not clusterin in control brains, was partially resistant to protease digestion in Western blots of total brain homogenates immunostained with anti-clusterin antibodies, which were processed in parallel with Western blots to PrP, without and with pre-incubation with proteinase K. Protein aggregation was analyzed in brain homogenates subjected to several solvents. PrP was recovered in the deoxycholate fraction in control and CJD cases, but in the SDS fraction only in CJD, thus indicating differences in PrP solubility between CJD and controls. Clusterin was recovered in the cytosolic, deoxycholate and SDS fraction in both CJD and control cases, but only clusterin from CJD was recovered in the urea-soluble fraction and, especially, in the remaining pellet. These findings demonstrate the capacity of clusterin to form aggregates and interact with PrPRES aggregates. The implications of this property are not known, but it can be suggested that clusterin participates in PrP clustering and sequestration, thus modifying PrP toxicity in CJD.

Adenosine A2A Receptors are Up-regulated in Pick’s Disease Frontal Cortex

Adenosine A2A receptors (A2AR) are highly expressed in striatum. However, they are also present in extrastriatal structures. A2AR were studied in post‐mortem human frontal cortex from Pick’s disease (PiD) and age‐matched non‐demented controls by radioligand binding assays, Western‐blotting, real‐time PCR and adenylyl cyclase activity determination. Saturation binding assay using [3H]ZM 241385, a selective A2A antagonist, as radioligand revealed a significant increase in total adenosine A2AR numbers (Bmax) in frontal cortex from PiD samples (191% of control Bmax), suggesting up‐regulation of this receptor. A significant increase in the level of A2AR was also detected by Western‐blotting. Furthermore, expression of mRNA coding A2AR determined by quantitative real‐time PCR was enhanced. In agreement, stimulation of adenylyl cyclase by CGS 21680, a selective A2A receptor agonist, was significantly strengthened. Up‐regulation of A2B receptors and their corresponding mRNA was also observed. These results show that A2A adenosine receptor/adenylyl cyclase transduction pathway is up‐regulated and sensitized in frontal cortex brain from PiD.

Abnormal group I metabotropic glutamate receptor expression and signaling in the frontal cortex in Pick disease.

Group I metabotropic glutamate receptors (mGluR1) regulate synaptic transmission through the stimulation of phospholipase Cβ1 (PLCβ1) and then by the activation of protein kinase C (PKC). Considering these properties, it is conceivable that major cortical functional deficits may be attributed to abnormal mGluR processing and signaling. The present work examines mGluRI expression and signaling in the frontal cortex (area 8) of 3 cases with Pick disease (PiD), a neurodegenerative disease with abnormal phospho-tau accumulation, in comparison with 3 age-matched controls by means of glutamate binding assays, enzymatic activity, gel electrophoresis and Western blotting, solubility and immunoprecipitation assays, and confocal microscopy. Reduced expression levels of PLCβ1 and reduced PLCβ1 activity have been found in PiD. The expression levels of the nonrelated phospholipase PLCγ, a substrate of tyrosine kinase, are also reduced in PiD. This is accompanied by a marked decrease in the expression of cPKCα and increased expression of the inner band (76 kDa) of the nPKCδ doublet at the expense of a decrease of the phosphorylated (active) form (78 kDa). In contrast, L-[3H]glutamate-specific binding to mGluRs is augmented in PiD cases, mainly because of the higher mGluR1 and mGluR5 expression levels detected. No modifications in PLCβ1 solubility have been observed in PiD and no interactions between PLCβ1 and tau have been demonstrated in diseased and control cases. Moreover, double-labeling immunofluorescence and confocal microscopy have shown no colocalization of phospho-tau (AT8 antibody) and PLCβ1 in phospho-tau inclusions, including Pick bodies. These results demontrate for the first time abnormal mGluR signaling in the cerebral cortex in PiD and selective vulnerability of phospholipases and PKC to PiD.

Adenosine A1 receptor protein levels and activity is increased in the cerebral cortex in Creutzfeldt-Jakob disease and in bovine spongiform encephalopathy-infected bovine-PrP mice.

Prion diseases are characterized by neuronal loss, astrocytic gliosis, spongiform change, and abnormal protease-resistant prion protein (PrPres) deposition. Creutzfeldt-Jakob disease (CJD) is the most prevalent human prion disease, whereas scrapie and bovine spongiform encephalopathy (BSE) are the most common animal prion diseases. Several candidates have been proposed as mediators of degeneration in prion diseases, one of them glutamate. Recent studies have shown reduced metabotropic glutamate receptor/phospholipase C signaling in the cerebral cortex in CJD, suggesting that this important neuromodulator and neuroprotector pathway is attenuated in CJD. Adenosine is involved in the regulation of different metabolic processes under physiological and pathologic conditions. Adenosine function is mediated by adenosine receptors, which are categorized into 4 types: A1, A2A, A2B, and A3. A1Rs are G-protein-coupled receptors that induce the inhibition of adenylyl cyclase activity. The most dramatic inhibitory actions of adenosine receptors are on the glutamatergic system. For these reasons, we examined the levels of A1Rs in the frontal cortex of 12 patients with CJD and 6 age-matched controls and in BSE-infected bovine-PrP transgenic mice (BoPrP-Tg110 mice) at different postincubation times to address modifications in A1Rs with disease progression. A significant increase in the protein levels of A1Rs was found in the cerebral cortex in CJD and in the murine BSE model at advanced stages of the disease and coincidental with the appearance of PrPres expression. In addition, the activity of A1Rs was analyzed by in vitro assays with isolated membranes of the frontal cortex in CJD. Increased activity of the receptor, as revealed by the decreased forskolin-stimulated cAMP production in response to the A1R agonists cyclohexyl adenosine and cyclopentyl adenosine, was observed in CJD cases when compared with controls. Finally, mRNA A1R levels were similar in CJD and control cases, thus suggesting abnormal A1R turnover or dysregulation of raft-associated signaling pathways in CJD. These results show, for the first time, sensitization of A1Rs in prion diseases.

Metabotropic glutamate receptor/phospholipase C pathway : A vulnerable target to creutzfeldt-jakob disease in the cerebral cortex

Glutamate is the main excitatory neurotransmitter in the cerebral cortex. Altered glutamatergic transmission has been suggested as having a central role in many neurodegenerative diseases. Metabotropic glutamate receptors (mGluRs) are coupled to intracellular signal transduction via G proteins, and they mediate slower responses than ionotropic glutamate receptors. Group I mGluRs are positively coupled to phospholipase C beta1 (PLCbeta1). Creutzfeldt-Jakob disease (CJD) is a human transmissible spongiform encephalopathy associated with a dysfunction in the membrane glycoprotein PrP which is converted into an abnormal isoform, with a predominant beta-sheet structure, that is pathogenic and partially resistant to protease digestion. Proteins associated with the signal transduction of group I mGluRs were examined in the frontal cortex (area 8) of 12 cases with sCJD and four age-matched controls, by means of gel electrophoresis and Western blotting of total homogenates. Densitometric analysis of the bands demonstrated decreased expression levels of PLCbeta1 and PLCgamma, a non-related phospholipase which is a substrate of tyrosine kinase, in CJD cases when compared with controls. Novel protein kinase C delta (nPKCdelta) has also been found to be significantly decreased in CJD cases. However, no modifications in mGluR1 cPKCalpha expression levels are found in CJD when compared with controls. No modifications in PLCbeta1 solubility in PBS-, deoxycholate- and sodium dodecylsulphate-soluble fractions have been observed in CJD when compared with controls. Finally, no interactions between PLCbeta1 and PrP, as revealed by immunoprecipitation assays, have been found in CJD and controls. The present results show, for the first time, reduced expression levels of phospholipases, particularly PLCbeta1, which may interfere with group I mGluR signaling in the cerebral cortex in CJD. These abnormalities are not the result of abnormal PLC solubility or interactions with PrP. Selective involvement of group I mGluRs may have functional effects on glutamatergic transmission modulation and processing in CJD.

Expression levels of adenosine receptors in hippocampus and frontal cortex in argyrophilic grain disease.

Expression of adenosine receptors of the A1, A2A and A2B type has been examined in the post-mortem frontal cortex and hippocampus in argyrophilic grain disease (AGD), a tauopathy affecting the hippocampus but usually not the frontal cortex, in an attempt to learn about the modulation of the adenosine pathway in this disorder. Significant increased levels of A1, but not of A2A and A2B, have been observed in AGD in the hippocampus but not in the frontal cortex, when compared with age-matched controls. This is accompanied by increased levels of adenylyl cyclase (AC), an effector of A1, and by increased (although not significant) percentage of inhibition of forskolin-stimulated AC by the A1 agonist cyclohexyladenosine in the hippocampus in AGD. These findings indicate sensitization of A1/AC in the hippocampus in AGD, and support a putative activation of the A1/AC pathway that may facilitate protection of this preferentially involved region in AGD.

Up-regulation of adenosine A1 receptors in frontal cortex from Pick's disease cases.

The adenosine A1 receptor (A1R)–adenylyl cyclase (AC) pathway was studied in post‐mortem human frontal and occipital cortex from Picks disease (PiD) cases and age‐matched nondemented controls. In frontal cortex, the main brain area affected in PiD, A1Rs, determined by radioligand binding, Western blotting and real‐time PCR assays, were significantly increased in PiD samples, suggesting up‐regulation of this receptor. AC activity was determined in basal and stimulated conditions via stimulatory guanine nucleotide binding proteins (Gs) using GTP, or directly with forskolin. Basal AC activity was reduced in brains from PiD cases. This agrees with the decrease in AC type I (AC I) level detected by Western blotting. However, inhibition of forskolin‐stimulated AC activity by a selective A1R agonist was significantly increased in brains from PiD. In occipital cortex, adenosine A1R numbers were similar in control and PiD cases, and no significant differences were found in A1R‐mediated AC inhibition. These results show that the adenosine A1R–AC transduction pathway is specifically up‐regulated and sensitized in frontal cortex brain in PiD.